Our long-term objectives are: 1) to determine the primary sequence and chromatin configuration of a mammalian chromosomal origin of replication; 2) to determine the molecular mechanisms by which chromosomal origins initiate replication at defined times during the S period; and 3) to determine the physical disposition of a mammalian chromosomal replicon in the nucleus during the replication process. Our laboratory has cloned a large part of the amplified dihydrofolate reductase (DHFR) replicon from a methotrexate-resistant CHO cell line. One of the recombinant cosmids (S21) contains the presumptive origin of replication, as defined by in vivo labelling studies on synchronized cells. The specific aims of the proposed project are: 1) to detrmine more precisely the location of the origin within the region defined by cosmid S21, utilizing cloned probes to analyze the earliest replication intermediates; 2) to define the minimum sequnce required for initiation, by testing subclones from S21 for their ability to replicate autonomously in CHO cells after introduction by spheroplast fusion; 3) if the studies above indicate the presence of a fixed origin in the DHFR domain, we will determine the primary sequence of the minimal origin; this data may suggest possible secondary structure, or the presence of enhancer or promoter elements, and the sequence can eventually be compared to that of origins isolated from other amplified replicons; 4) to determine in nuclease sensitivity studies whether the initiation locus has an altered configuration, and, if so, whether this configuration changes during the cell cycle; 5) to determine whether the initiation locus changes its relationship to fixed nuclear structures during the replication process, by utilizing radioactive probes to analyze nuclear matrix/DNA halo structures and sectioned nuclei isolated at different times during the cell cycle; the latter two specific aims are designed to provide insight into the replication process regardless of whether initiation in the DHFR domain begins at random or at fixed positions within the region defined by cosmid S21. If we are able to isolate an origin of replication from the DHFR domain in the studies outlined above, and if time permits, we will utilize the same approach to isolate at least one other origin of replication from a cell line that has amplified the transfected DHFR gene in a new chromosomal location.